Abstract
Publisher Summary The functional significance of the mRNA poly(A) tail has been debated over the years and several different functions have been assigned to it—for example, as a structural element influencing the translational efficiency, the mRNA half-life, and/or the intracellular transport of mRNAs. Poly(A) removal is an important step during mRNA decay and it has been established that mRNA degradation is initiated by degrading the mRNA poly(A) tail. This chapter describes the purification of the 54-kDa PARN nuclease isoform from calf thymus cell-free extracts. The same protocol can be used to purify PARN from HeLa cell-free extracts. A simple procedure to isolate the HeLa cell activity has been described. This procedure provides approximately 3 μ g of a homogeneously purified preparation of the 54-kDa active fragment of the PARN nuclease. Starting with 3 kg of calf thymus the recovery of 3 μ g is not impressive; however, the resulting preparation retains all the important properties of the nuclease activity. It is poly(A) specific, oligomeric, and processive, and it interacts with the Y-end mRNA cap structure. Future experiments with recombinant PARN will rely on a comparison with the native PARN activity.
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